1 In the proposal we develop dual imaging and targeting photosensitizing agents that are specifically designed 2 as substrates for phosphatidylcholine-specific phospholipase C (PC-PLC). PC-PLC is overexpressed and 3 highly active in breast cancer and is present on the outer leaflet of the plasma membrane. The highest level of 4 PC-PLC expression has been detected in aggressive treatment-resistant triple negative breast cancer cells 5 with high proliferative potential. The selective compartmentalization and simultaneous activation of PC-PLC in 6 cancer cells suggest that this metabolic enzyme may be useful as a target for diagnosis and treatment of 7 breast cancer, including triple negative type with high metastatic potential. Recently, we have successfully 8 designed, synthesized and tested a near infrared fluorescent self-quenched phospholipid probe that displays 9 high sensitivity and selectivity to PC-PLC. Our probe is dark in the native state, but becomes fluorescent after 10 hydrolysis by PC-PLC. The fluorophore contained in this probe, pyropheophorbide a, can also act as a 11 photosensitizer and thus the probe can function as both an imaging and photodynamic therapy (PDT) agent. 12 Thus the goal of this proposal is to use PC-PLC as a novel target for detection and PDT therapy of breast 13 cancer. Since PC-PLC probes are highly lipophilic, we have developed lipid-based nanoparticles (LNPs) to 14 deliver the probes to the tumor site in vivo. The composition of the LNPs will be optimized to fabricate small- 15 sized particles with reduced non-specific adsorption and high probe payload. When the probes intercalate into 16 the cancer cell membrane, PC-PLC will induce specific cleavage, restoring fluorescence and phototoxicity. We 17 will employ a panel of human breast cancer cell lines and examine PC-PLC probe uptake and activation, the 18 probe induced enzyme internalization, determine subcellular probe localization and measure phototoxicity. We 19 will compare the level of PC-PLC activity in breast cancer cell lines, including triple negative cells, with cells 20 from normal tissue. This difference will provide the basis for imaging and selective damage of breast cancer 21 cells during PDT in vivo. Human breast cancer cells will be engrafted in athymic nude mice to study the 22 imaging potential of our PC-PLC probe in vivo. We will measure probe delivery to the tumor site, kinetics and 23 sensitivity of probe activation and probe biodistribution. We will employ an immunocompetent syngeneic breast 24 cancer mouse model to compare the phototoxic effect of our PC-PLC targeted probes with untargeted probe 25 on the progression of breast tumors with high metastatic potential. The heterogeneity of PC-PLC expression 26 will be evaluated on these tumors ex vivo. Since the activated probe selectively accumulates in the breast 27 cancer cell plasma membrane, PDT could induce a tumor-specific immune response, reducing tumor 28 recurrence and metastasis. The completion of this project will provide molecular oncology information 29 regarding abnormal expression and activity of PC-PLC in different breast cancer cells as well as clinical 30 oncology information about the potential of PC-PLC probes as in vivo imaging and PDT theranostic agents.